1. Field of the Invention
The present invention relates generally to the field of cellular injury. More specifically, the present invention relates to the uses of collagen IV in promoting recovery of cellular functions following cellular injury.
2. Description of the Related Art
The mesh-like basement membrane (BM) provides structural support and influences the growth, function, and survival of many cell types in most organ systems (15). Collagens are extracellular matrix (ECM) proteins that form the renal tubular basement membrane with other extracellular matrix proteins, such as laminin and heparan sulfate proteoglycans (29). The most abundant type of collagen in the basement membrane of the glomerulus and renal tubules is collagen IV, a globular, non-fibrillar protein. This characteristic distinguishes it from collagen I, the major fibrillar component of connective tissues and the second most abundant extracellular matrix protein in the proximal tubular basement membrane (12, 21).
Collagen IV forms a triple-helical monomer that consists most often of two α1(IV) chains and one α2(IV) chain or three α1(IV) chains (42,14). The collagen IV chains α3(IV), α4(IV), α5(IV), and α6(IV) have been identified and can associate in various combinations (15, 20). However, these isoforms have not been detected in the human proximal tubule or in primary cultures of rabbit renal proximal tubular cells (12, 29). Except in rodents, their renal expression appears to be limited to the distal tubular basement membrane and the glomerular basement membrane, where they have been implicated in the development of Goodpasture and Alport syndromes and diffuse leiomyomatosis (27, 29, 11, 15, 19, 20). Using functional analyses of cell-matrix interactions, collagen IV has been shown to play a crucial role in tubular function and kidney development (31). Because collagen IV is an important anchorage substrate for many cell types, especially in the kidney, the regulation of collagen IV synthesis and degradation plays an important role in cell function, growth, migration, and organ remodeling (15).
Under conditions of ischemia or after acute chemical exposures, renal epithelial cells may die or detach from the extracellular matrix and slough into the tubular lumen. Here they may aggregate with other sloughed cells, forming casts that cause tubular obstruction. Cells that do not die or become detached from the extracellular matrix are thought to dedifferentiate, proliferate, and migrate to denuded areas of the tubule, thus replacing the sloughed cells. The cells of the newly lined tubule may then differentiate, promoting the return of normal tubular function and overall renal function (1). The roles of collagens and other extracellular matrix proteins in renal cell survival, migration, and function have been examined (4). Surprisingly, few reports exist regarding the role of collagens in cellular repair and regeneration, although proliferation, migration, and return of normal functions do contribute to renal regeneration following injury (49).
Ascorbic acid is known to prevent the effects of scurvy, a disease characterized by defective connective tissue resulting from decreased collagen synthesis (40). In post-translational processing mechanisms, ascorbate acts as an essential iron reducing cofactor in the production of collagens, specifically in the hydroxylation of susceptible proline and lysine residues in procollagen α chains. These hydroxylation reactions are catalyzed by prolyl and lysyl hydroxylases, respectively, and are necessary for the proper folding of procollagen triple helices, as well as other post-translational modifications, including glycosylation and monomer crosslinking (9,24). Insufficiently hydroxylated procollagens have been shown to accumulate intracellularly, be deposited much more slowly, and be targeted for rapid degradation both intracellularly and extracellularly (17, 18, 42). Ascorbic acid also is known to promote the synthesis of both fibrillar and non-fibrillar collagen types in an array of cell types in vitro (10, 13, 33, 45). In addition, ascorbate has been suggested to act pretranslationally by stimulating mRNA expression of multiple collagen types in various culture systems, independent of its role as an enzymatic cofactor (6, 14, 32, 41, 46). Ascorbic acid has been implicated as an important mediator of cell growth and differentiation in a variety of cell types, through its effects on collagen synthesis and deposition (2). Through mechanisms unrelated to extracellular matrix production, ascorbic acid has been shown to both stimulate and inhibit cell proliferation depending on ascorbate concentration and cell type (48, 7, 16). Previous studies have demonstrated that ascorbic acid promotes increased cell growth and density, and improvement of key physiological functions including brush border enzyme activity, basal oxygen consumption, and Na+-K+-ATPase activity in primary cultures of rabbit renal proximal tubular cells (RPTC) (36).
The halocarbon conjugate S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is a model toxicant that produces renal proximal tubular cell necrosis and acute renal failure (23). Primary cultures of rabbit renal proximal tubular cells sublethally injured by S-(1,2-dichlorovinyl)-L-cysteine neither proliferate nor repair physiological functions (38). In those experiments, renal proximal tubular cells were grown under physiological concentrations of all culture media supplements including 50 μM L-ascorbic acid 2-phosphate (AscP). However, upon addition of pharmacological concentrations of L-ascorbic acid 2-phosphate (500 μM), renal proximal tubular cells exposed to S-(1,2-dichlorovinyl)-L-cysteine were able to proliferate and repair physiological functions, although L-ascorbic acid 2 phosphate provided no protective effect during injury. In addition, pharmacological concentrations of ascorbic acid were shown to stimulate collagen IV synthesis and deposition in uninjured renal proximal tubular cells (39).
Cellular integrins are heterodimeric transmembrane receptors that provide a means for anchorage to extracellular substrates as well as two-way communication between the intracellular and the extracellular environments (Molitoris and Marrs, 1999; Ruoslahti and Engvall, 1997; Schoenwaelder and Burridge, 1999). Activation and clustering of integrins upon binding to extracellular matrix proteins initiate focal adhesion formation and the activation of cytoskeletal signaling cascades involved in cell growth, proliferation, migration, differentiation, and gene expression (Molitoris and Marrs, 1999; Schoenwaelder and Burridge, 1999, Zuk et al., 1998). In addition to binding to extracellular matrix substrates and mediating cytoskeletal signaling, integrins also are known to influence the formation and composition of the extracellular matrix (Gotwals, et al., 1996; Riikonen et al., 1995). In renal proximal tubular cells, integrins and other proteins, such as Na+/K+-ATPases, are localized to the basal membrane, where cells interact with the extracellular matrix as well as neighboring cells. These functions are in contrast to those of the apical membrane, where distinct physiological processes such as Na+-dependent glucose and amino acid transport take place. The cellular polarity derived from the distinct functions carried out at separate membrane regions supports, and is critical for, proper renal tubular function (Bush et al., 2000).
The renal tubular basement membrane is composed mainly of collagens, laminins, and heparan sulfate proteoglycans (Furness, 1996, Miner, 1999). The most abundant type of collagen in the basement membrane of the glomerulus and renal tubules is collagen IV, a globular, non-fibrillar protein (Furness, 1996). The binding of integrins to collagens and other extracellular matrix proteins is determined largely by the combination of α and β integrin subunits that form the functional heterodimer. At least eight β subunits and 17 α subunits have been identified to date, and they associate non-covalently to form more than 20 heterodimers with various signaling and substrate binding properties (Kreidberg and Symons, 2000). Cells most often utilize the integrin heterodimers α1β1 and α2β1 to bind collagen IV, and the importance of signals derived from collagen-binding integrins (CBIs) in normal cellular activities have been studied (Gardner et al., 1996; Knight et al., 1998; Kuhn and Ebel, 1994).
In cases of acute renal failure resulting from chemical exposure or ischemia, tubular epithelial cells may lose polarity, as characterized by decreased localization of integrins in the basal membrane and their redistribution throughout the plasma membrane (Goligorsky and DiBona, 1993; Lieberthal et al., 1997;
Molitoris and Marrs, 1999; Zuk et al., 1998). Loss of cellular polarity results in cellular disorientation, decreased renal tubular function, and cell death and/or detachment from the tubular basement membrane (Frisch and Ruoslahti, 1997; Goligorsky and DiBona, 1993; Molitoris and Marrs, 1999; Tang et al., 1998). Sublethally injured cells that do not die or become detached from the basement membrane are thought to repair and/or dedifferentiate, proliferate, migrate to denuded areas of the tubule, differentiate, and promote the return of normal renal function (Abbate and Remuzzi, 1996; Molitoris and Marrs, 1999). The effects of cell injury on integrin localization and renal cell polarity have been investigated, but their importance in tubular regeneration following injury is not well understood (Goligorsky and DiBona, 1993; Lieberthal et al., 1997; Kreidberg and Symons, 2000; Molitoris and Marrs, 1999; Zuk et al, 1998).
Studies have examined the mechanisms of renal tubular cell regeneration using the model nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine to produce sublethal injury in primary cultures of rabbit renal proximal tubular cells. Exposure to S-(1,2-dichlorovinyl)-L-cysteine to produce approximately 50% cell death and loss caused the irreversible inhibition of key physiological functions, including mitochondrial function, active Na+ transport, and Na+/K+-ATPase activity in the remaining sublethally-injured renal proximal tubular cells (Nowak et al., 1999). However, addition to the culture media of L-ascorbic acid-2-phosphate at pharmacological concentrations promoted proliferation and repair of physiological functions in S-(1,2-dichlorovinyl)-L-cysteine-injured renal proximal tubular cells (Nowak et al., 2000).
Thus, the prior art is deficient in novel uses of collagen IV. The present invention fulfills this long-standing need and desire in the art.